IkappaB kinase (IKK) catalytic subunits play a key role in cytokine-mediated nuclear factor (NF)-kappaB signaling, and a loss of NF-kB function appears to inhibit inflammation and oncogenesis. Manumycin A, a natural epoxyquinoid compound, is a potent and selective farnesyltransferase inhibitor with antitumor activity. Our recent findings indicate that treatment with manumycin A resulted in a rapid, selective and potent inhibition of TNF alpha-stimulated IKK activity in a number of cell lines and a primary culture of rat hepatocytes. Unexpectedly, other classes of farnesyltransferase inhibitors had no inhibitory effect. To identify the molecular mechanisms of manumycin A action, we transfected human HepG2 hepatoma cell line with IKKalpha and IKKbeta constructs and demonstrated direct inhibition of IKK activity with concomittant formation of stable homotypic IKKbeta dimers in the presence of manumycin A. A number of Cys-> Ala point mutants of IKKbeta were generated to investigate the possibility that IKKbeta covalent dimerization results from nucleophilic attack on the epoquinoid core of manumycin A. Cells expressing IKKbeta mutated in the activation loop at Cys-179 exhibited similar dimer formation, whereas double substitution of Cys-662 and ?716 conferred protection against homotypic dimerization by manumycin A. IKK interaction with the adaptor protein IKKgamma/NEMO was also disrupted in manumycin A-treated cells. Thus, manumycin A plays important regulatory function in IKK signaling through pathways distinct from its role as farnesylation inhibitor. We are currently using an in vivo approach to investigate the hypothesis that manumycin A affects the activation of IKKbeta, a major contributor to inflammation-induced insulin resistance. Injection of various concentrations of manumycin A has been carried out in murine B16F10 melanoma implanted s.c. in the back of several mice. The tumor masses were excised, weighted and are currently being processed for Western blot, in vitro IKK activity and EMSA (NF-kappaB) studies. It is anticipated that our results will demonstrate that IKKbeta is a direct target of manumycin A in vivo. Future plans include the study of the effect of manumycin A in the regulation of insulin responsiveness in physiological and pathophysiological pro-inflammatory states such as diet-induced obesity and aging.